Significant progress has been achieved in the management of adult acute leukemia. However, long term disease free survival is currently achieved in less than 50% of patients. Optimal therapy has not yet been defined and stratification of therapy according to prognostic factors is only now beginning to be addressed. Acute leukemia (AL) is an uncommon form of malignancy affecting approximately five persons per 100,000 in the U.S. annually with a 90% mortality rate within one year of diagnosis. Although incidence of leukemia over all has been stable for the last 30 years, the increasing age of the population will presumably result in a greater number of cases being reported.
On the other hand, malignant lymphomas are the seventh most common cause of death from cancer in the United States. In 1992, approximately 41,000 new cases were diagnosed, and there were approximately 19,400 deaths from the disease. The incidence of lymphocytic lymphomas is increasing each year; the 50% increase in incidence between 1973 and 1988 reported by the American Cancer Society was one of the largest increases reported for any cancer. A large portion of the increase has been attributed to the lymphocytic lymphomas developing in association with acquired immunodeficiency syndrome (AIDS).
In the treatment of acute myelogenous leukemia (AML) in adults corticosteroids, methotrexate, 6-mercaptopurine and vincristine were used, initially as single agents and subsequently in combination. The emergence of ara-C as the major single agent in the management of AML in the late 1960s converted this incurable disease to a potentially curable condition. The subsequent development of anthracyclines, such as daunorubicin, doxorubicin (Adriamycin), rubidazone and idarubicin allowed new combinations to be developed. The vinca alkaloids, alone or in combination with corticosteroids, and epidophyllotoxins, methotrexate, 6-mercaptopurine, cyclophosphamide and L-asparaginase have only minor activity against adult AML. Most regimens in adult AL incorporate vincristine, corticosteroids, prednisone or dexametlhasone, anthracyclines, and L-asparaginase.
The predominant treatments for AL have been in place for 5 to 15 years. No major class of drugs for the management of AL has been discovered. The allogenic and autologous marrow transplant program with monoclonal purging and chemotherapy purging have been applied with no major difference in response or survival rates.
The treatment approach to a particular patient with lymphocytic lymphoma is determined by the tumor histology, the stage of disease, and the physiologic status of the patient. The most active chemotherapy programs for intermediate-grade lymphomas include therapy using different combinations, dosages and schedules of cyclophosphamide, doxorubicin, vincristine, procarbazine, prednisone, bleomycin and methotrexate. Development of new drugs that are selective for particular subsets of leukemia and lymphoma patients is of the highest priority.
Second only to lung cancer as a cause of cancer deaths, prostate cancer has become the most common cancer among American men. It was predicted that 316,000 new cases of prostate cancer would be diagnosed in 1996, causing 40,000 deaths. Potential prostate cancer chemopreventive agents under consideration are diverse with respect to source, chemical structure and physiological effects. The candidate drugs under development for treatment of prostate cancer must demonstrate long-term safety and minimum toxicity after long term administration. Retinoids such as lycopene and 4-hydroxyphenretiminide and difluoromethyl ornithine (DFMO) are being evaluated in patients scheduled for radical prostatectomy. Androgen deprivation may also have a role in chemoprevention. Other approaches under study include the use of vitamin D analogs, .alpha.-tocopherol and dietary restriction of fat. Conventional chemotherapeutic agents used for hormonally relapsed prostate cancer are doxorubicin, ketonazole, cyclophosphamide, prednisolone, estramustine, vinblastine, paclitaxel, mitoxantrone, either alone or in combination.
The vast majority of ovarian cancers are epithelial in origin and these account for more than 90% of the estimated 26,700 new cases of ovarian cancers that were diagnosed in the United States in 1996. Ovarian cancer is the leading cause of death from a gynecologic cancer. From 1970 to 1991, survival rates for patients with ovarian cancer have increased from 36% to 44%. This improvement in survival rate is in large part due to the development of curative platinum-based chemotherapy for patients. The generally accepted treatment for patients with either stage III or IV (advanced-stage) ovarian cancer has been similar: cytoreductive surgery when feasible, followed by chemotherapy. Chemotherapeutic agents from a wide variety of different classes have been shown to produce responses in patients with ovarian cancer. Before the demonstration of marked activity of paelitaxel in ovarian cancer, platinum compounds were considered to be the most active agents in this disease. Both paclitaxel and docetaxel have been demonstrated to have activity in platinum-resistant patients. Gemcitabine, a pyridine antimetabolite, has been shown in phase II trials in Europe to be an active agent.
Under the auspices of a National Cooperative Natural Product Drug Discovery Group supported by the National Cancer Institute, the potential antitumor activity of approximately 2500 extracts derived from globally collected plants was evaluated in a panel of enzyme based assays and in a battery of cultured human tumor cell lines. One such extract, prepared from the stem bark of Ziziphus mauritiana Lam. (Rhamnaceae), displayed selective cytotoxicity against cultured human melanoma cells (Nature Medicine, Pisha et al., Vol. 1, No. 10, pages 1046-1051, October 1995; WO 96/29068). As a result of bioactivity guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a mclanoma-speciflic cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumor growth was completely inhibited without toxicity. As judged by a variety of cellular responses, antitumor activity was mediated by the induction of apoptosis. On page 1047 of Nature Medicine, Vol. 1 (10), 1995 it is stated that growth of human cancer cell types other than melanoma were recalcitrant to treatment with betulinic acid. It is also disclosed on page 1049 that although initial reports reported betulinic acid as active against the Walker 256 murine carcinosarcoma tumor system, L1210 murine lymphocytic leukemia model and P-388 murine lymphocyte test, these results were not reproduced in subsequent tests.
A number of triterpenoids, including betulinic acid, have several known medical applications including use as an anticancer drug. Anderson et al., in WO 95/04526, discuss derivatives of triterpenoids which have been used in cancer therapy, including their activity against polyamines which are required by cells to grow at an optimal rate. Some of these triterpenoids have been found to interfere with enzymatic synthesis of polyamines required for optimal cell growth, and thus inhibit the growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K. et al. Oncology 48 : 72-76, 1991). Thle anti-cancer activity of betulinic acid and some derivatives has been demonstrated using mouse sarcoma 180 cells implanted subcutaneously in nude mice (JP 87-301580). Choi et al have shown that betulinic acid 3-monoacetate, and betulinic acid methyl ester exhibit ED.sub.50 values of 10.5 and 6.8 .mu.g/ml, respectively, against P388 lymphocytic leukemia cells (Choi, Y-H et al., Planta Medica vol XLVII, pages 511-513, 1988). According to Choi, a compound is considered active in the P388 system if it has an ED.sub.50 of .ltoreq.4.0 .mu.g/ml.
Betulinic acid has been reported also to possess anti-inflammatory activity. The anti-inflammatory activity of betulinic acid is, at least in part, due to its capacity to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase (Somatsu, S. et al., Skin and Urology 21: 138, 1959 and Inoue, H., et al., Chem Pharm Bull. 2: 897-901, 1986).
Betulinic acid and dihydrobetulinic acid acyl derivatives have been found to have potent anti-HIV activity (WO 96/39033). Anti-HIV assays indicated that 3-O-(3', 3'-diimethylsuccinyl)-betulinic acid and dihydrobetulinic acid both demonstrated extremely potent anti-HIV activity in acutely infected H9 lymphocytes with EC.sub.50 values of less than 1.7.times.10.sup.-5 .mu.M, respectively.
Thus, betulinic acid and its derivatives have been shown to possess several medicinal properties including anticancer activity. But apart from the conclusive evidence of its anti-melanoma activity, it has not so far been shown to possess anticancer activity on other cancers. We report here for the first time anti-leukemia and anti-lymphoma activity of betulinic acid and its derivatives with ED.sub.50 values in the range of approximately 0.5 to 4.0 .mu.g/ml. Further we also report for the first time anti-prostate cancer activity of betulinic acid and its derivatives with ED.sub.50 values in the range of approximately 0.6 to 6.8 .mu.g/ml. Activity against lung cancer is also demonstrated. Betulinic acid derivatives have an ED.sub.50 value in the range of 0.4 to 8.1 .mu.g/ml against ovarian cancer.